Mixed anhydride method of preparing peptides

ABSTRACT

AN IMPROVED PROCESS IS DESCRIBED FOR PREPARING PEPTIDES BY THE MIXED ANHYDRIDE METHOD. THE IMPROVEMENT CONSISTS IN USING CERTAIN SPECIAL AMINES WHICH HAVE AT LEAST ONE METHYL GROUP ATTACHED TO NITROGEN, AND AN ELECTRONEGATIVE GROUP BETA TO THE NITROGEN. THE AMINES MAY BE USED IN EXCESS UP TO 150% AND THE PEPTIDE IS SYNTHESIZED WITH NEGLIGIBLE RACEMIZATION OF THE STEREO ISOMERS OF THE CONSTITUENTS WHICH ARE JOINED TO FORM THE PEPTIDE.

United States Patent U.S. Cl. 260-1125 4 Claims ABSTRACT OF THEDISCLOSURE An improved process is described for preparing peptides bythe mixed anhydride method. The improvement consists in using certainspecial amines which have at least one methyl group attached tonitrogen, and an electronegative group Beta to the nitrogen. The aminesmay be used in excess up to 150% and the peptide is synthesized withnegligible racemization of the stereo isomers of the constituents whichare joined to form the peptide.

RELATED APPLICATIONS This application is a continuation-in-part of mycopending application 622,049, filed Mar. '10, 1967, now abandoned.

An application of George Washington Anderson, Francis Marc Callahan, andJoan Estelle Zimmerman, Ser. No. 757,481, filed Sept. 4, 1968, and nowabandoned, which is a continuation-in-part of their co-pendingapplication Ser. No. 727,704, filed May 8, 1968, and now abandoned,describes synthetic peptides having thyrocalcitonin activities, and bothof these latter applications are owned by the assignee of the presentinvention.

BACKGROUND OF THE INVENTION Synthesis of peptides using the mixedanhydride procedure is well established and valuable. A review of theprocedure has been presented by A'lbel'tson, Noel F., as Chapter 4,Synthesis of Peptides with Mixed Anhydrides, pages 157-355, OrganicReactions, vol. 12, John Wiley and Sons, Inc., New York, London, 1962.

Essentially the procedure consists of the two steps of (l) forming amixed anhydride by reacting an a-acylamino acid or a u-acylamino peptidewith an alkyl chloroformate in the presence of a tertiary amine base,and then (2) reacting the resulting mixed anhydride with an amino acidderivative or peptide derivative having a free amino group to form apeptide:

3,640,991 Patented Feb. 8, 1972 wherein X, Y, R R and R are ashereinafter defined, Z is a protective group and A and A the residueremaining of an amino acid or peptide.

The process has proved to be difiicult and critical, because unless thebases used, such as tertiary amines, are practically in equi-molecularproportions, racemization of the stereo isomers of the peptides or aminoacids that are joined in forming the new peptide may occur. As for manypurposes, racemized peptides are either useless, where this has causedthe loss of physiological activity, or are seriously affected, whichoccurs when the racemization is only partial. Many peptides which are tohave physiological use are in the laevo form, but certain substances, 2such as pantothenic acid, are difiicult in that they are active only inthe dextro form.

The critical necessity of using the tertiary amines in substantiallyequi-molecular proportions is a very serious practical drawback. Itmakes the process extremely critical 3 and in some cases reduces theeffectivness of the process, as in many instances an excess of thetertiary amine can be used and the reaction proceeds either more rapidlyor to a greater degree of completion.

Some peptide esters, particularly esters of dipeptides, formdiketopiperazines very readily. When employing such peptide esters inmixed anhydride synthesis, it is of course desirable to avoiddiketopiperazine formation, and this avoidance can be achieved by addingthe peptide ester in the form of its salt, rather than as a free base,to the 40 mixed anhydride mixture containing an excess (about 100%) ofone of the defined tertiary amines of the invention. In this way, as thepeptide ester salt is liberated as free base by the excess amine duringthe addition, it reacts immediately with the mixed anhydride, therebyforming desired product and avoiding diketopiperazine formation.

SUMMARY- OF THE INVENTION The present invention is based on thesurprising discovery that if certain special tertiary amines are used;namely those having at least one methyl group attached to the aminonitrogen, and an electro-negative group Beta to the amino group, theycan be used in excesses, preferably not greater than 150% excess, andthe process proceeds rapidly with excellent conversions and with noracemization. Examples of these special amines are those in which theelectronegative group is an atom of the 6th group of the Periodicsystem, such as N-methylmorpholine, or a group having a member of the5th group of the Periodic system, such as N,N'-dimethylpiperazine.Another illustration of an electro-negative group is one which involvesa triple bond. This can, for example, be a triple bond joining afi-carbon atom to another carbon atom, such asN,N,N',N-tetramethyldiaminobutyne-2. The triple bond can also be in aseparate group which is Beta to the amino nitrogen, for example,18-cyanoethyldimethyl amine. It is not known why these particularspecial types of tertiary amines can be used in excess with negligiblerisk of racemization, and it is not intended to limit the invention toany theory of why this surprising result occurs.

The mixed anhydride process, of which the present invention is animprovement, utilizes a chloroformate, as appears from the generaldescription of this process in the background of invention. Thechloroformate is a lower alkyl chloroformate, that is to say, not morethan 6 carbon atoms in the alkyl group, and the alkyl group maybe astraight chain, that is to say, forming a primary ester, or a chainbranching at the a-carbon atom, forming a secondary ester. However, itshould not be a tertiary alkyl group. Except for the use of the specialnew types of amines in excess, the general reaction procedure of themixed anhydride method is not significantly changed, which is anadvantage of the invention as new or critical techniques are notrequired.

As in all peptide syntheses of the mixed anhydride type, the amino groupof one of the two constituents entering in the reaction to form the newpeptide must be blocked. This is usually effected with an acyl group.The present invention is not limited to the use of any particular acylgroup, but acyl groups of the carbobenzoxy type or phthaloyl and certainsubstituted acylic groups, such as triphenylmethyl and trifiuoroacetyl,are preferred because they are very readily split off from the peptideform after the synthesis is complete. In the more specific descriptionsthese preferred types of blocking groups for the amino group of apeptide fragment or an a-amino acid will be described as typicalillustrations, but the invention is not limited thereto, and othergroups, such as acetyl or benzoyl and the like, are included althoughthey are less desirable because of the difficulty of removing them afterthe synthesis is complete.

In the present invention the general equation of a mixed anhydrideformation for use in the peptide synthesis may be written as follows:

X and Y designate the protective group, X is acyl and Y may be hydrogenif a mono-basic acyl is used or X and Y can be a divalent protectivegroup. Preferably, X is benzyloxycarbonyl, tertiarybutyloxycarbonyl,triphenylmethyl and trifluoroacetyl. R is lower alkyl excluding tertiaryesters, R is an ethyl group having an electronegative group on the )3carbon atom; R is lower alkyl, lower alkyl phenyl, or cycloalkyl of atleast 4 carbon atoms, or R and R may be parts of a heterocyclic ring,such as morpholine, N,N-dimethylpiperazine.

The peptides of the present invention are made up of the naturallyoccurring a-amino acids described, for example, by P. Karrer, OrganicChemistry, Second English addition, Elsevier Publishing Company, Inc.,New York, 1946. Among the naturally occurring amino acids may bementioned, for example, alanine, serine, aminobutyric acid, cystine,cysteine, methionine, norvaline, valine, norleucine, leucine,isoleucine, phenylalanine, tyrosine, dihydroxyphenylalanine,tryptophane, arginine, lysine, hydroxylysine, ornithine, aspartic acid,asparagine, glutamic acid, hydroxyglutamic acid, glutamine, glycine,histidine, thiolhistidine, proline, hydroxyproline, tyrosine,diiodotyrosine, thyroxine and threonine. Since these aretaminocarboxylic acids, they can be illustrated by the followingformula:

wherein R and R are the residue remaining of the atamino acids describedabove.

The reaction to prepare the mixed anhydrides of the present invention iscarried out at a temperature of from about 5 C. to about 20 C. Thereaction is preferably carried out under non-aqueous conditions in thepresence of the preferred solvent tetrahydrofuran. Other solvents may beused, such as dimethoxyethane, dimethylacetamide, ethyl acetate,dioxolane, triethylphosphate or 5,5-dimethyldioxolane; the use of suchother solvent being dependent upon the reactants. The reaction issubstantially complete in from one-half minute to 15 minutes.

The rest of the reaction follows the general equation of the mixedanhydride process which has been set out above. It should be noted thatthe process is not in any way limited to the peptides produced becausethe reaction is taking place only at one end of each of the two reactingconstituents; and if, as will usually be the case, one or both arethemselves peptides, the remainder of the peptide molecule is immaterialto the operation of the process. It is an advantage of the presentinvention that it may be used to produce peptides of any size, forexample those having thyrocalcitonin activity, such as thyrocalcitoninitself with 32 amino acids, or smaller fragments such as thosecontaining amino acids 1 to 16 of the thyrocalcitonin molecule. Otherpeptides which can be produced by the present invention includeoxytocin, insulin, vassopressin, and the like. Of course in thesynthesis of very large peptides, the synthesis usually proceeds in anumber of steps, smaller peptide fragments being first formed and joinedtogether or a single amino acid added. In general, the term peptide isused instead of polypeptide, although this latter is quite common in theliterature. Strictly speaking, any peptide is a polypeptide because itis made up of at least two amino acids. Also, it should be understoodthat in the present application, we are using the more generic term ofpeptide for compounds which may include certain compounds that are moreoften referred to as proteins. In general, a protein is simply apolypeptide of large molecular weight, usually over 10,000. As far asthe chemistry is concerned and as far as the reactions of the presentinvention are concerned, the two terms do not relate to different typesof compounds.

DESCRIPTION OF THE PREFERRED EMBODIMENT The following examples areillustrative of the use of the process of the present invention inpreparing a number of typical peptides or polypeptides. For the mostpart, the examples deal with the formation of peptides which are notusually used as such but are used in the further synthesis of peptideshaving physiological activity, such as those having thyrocalcitoninactivity. It should also be noted that the present invention need not beused in every step of a polypeptide synthesis. Sometimes it is used in anumber of steps and certain other steps may use other well known methodsof peptide synthesis. For instance, many of the examples which followcover a chain of synthesis which forms intermediate peptides that arefinally transformed into a physiologically active one. Often the laststep of such a process may involve preferably a different method ofcoupling, and it is a great advantage of the present invention that itcan be used in syntheses with other methods in certain steps, whichgives a great choice and flexibility to the protein chemist.

The examples in the present specification are illustrative of a fewpeptides which can be prepared by the process of the present invention,which is generally applicable to the enormous number of theoreticallypossible compounds. It is of interest to note that in most cases apeptide described in a particular example serves as an intermediate forfurther synthesis. Examples 1 8 to 107 illustrate a sequence of peptidesprepared by the present invention which finally result in the synthesesof peptides of sixteen, twenty-two, twenty-five and twenty-nine, aminoacids, respectively.

These four compounds have thryocalcitonin activity and when tested bythe conventional rat assay method, showed a reduction in serum calcium.These examples are taken in the main from the application of Anderson,Callahan and Zimmerman, Ser. No. 757,481, [filed Sept. 4, 1968 and nowabandoned. The new active peptide forms a part of the subject matter ofthe application referred to. It is not claimed in the presentapplication, which is directed primarily to a process, and the examplesare merely given to illustrate further peptide syntheses using thefeatures of the present application to finally produce a usefulphysiologically active preparation. A number of examples in addition tothe first seventeen, are set forth hereinbelow in order to showsyntheses of four polypeptides which have hormone activities. Because ofthe very large number of examples needed to set forth the various stepsin the long syntheses, and because most of the examples do notillustrate the process improvement of the present invention, thoseexamples which do not involve the essential features of the presentinvention are presented in abbreviated form, setting forth reactants andproducts and indicating the reaction conditions by brief notations.These examples use standard conditions for peptide synthesis inaccordance with the outline of general methods and conditions presentedhereinbelow. Formula and other abbreviations are defined in a glossarypreceding the examples. In some of the examples in addition to the firstseventeen, the synthesis features of the present invention areillustrated, and these examples are presented in full detail.

It should be noted that in many of the examples set forth hereinbelow inabbreviated form, activating groups such as the N-hydroxysuccinimide areused for the car boxyl end of peptides. This is not because the featuresof the present invention would not be usable in some or most of theseexamples, but because the examples represent the actual syntheses whichwere carried out.

The process features of the present invention are generally usable inpeptide syntheses but because of the Well known fact that the possiblenumbers of peptides and proteins is unlimited, only a moderate number oftypical illustrations are set forth in the examples it being understoodthat the in'vention is not limited to the production of the particularpeptides of the examples. It is an advantage of the present inventionthat the process is generally applicable in peptide syntheses and itseffectiveness opens up a very broad field of improved peptide syntheses.

The following glossary sets forth abbreviations used. in many of theexamples:

OMe-O-methyl M.A.-Mixed anhydride (usually standing for the method usedin a coupling reaction) HOAcAcetic acid EtOAc-Ethyl acetate Et O-Ether.Specifically, diethyl ether NMM-N-methylmorpholine TLC-Thin layerchromatography THFTetrahydrofuran SBzl-S-benzyl ONB-O-p-NitrobenzylBOC-t-Butyloxycarbonyl TFA-Trifluoroacetic acid or trifluoroacetate, asthe case may be.

6 I-IOSu-N-hydroxysuccinimide OSuN-hydroxysuccinimide esterZBenzyloxycarbonyl Z -Dibenzyloxycarbonyl Gl -G1utamic acid orglutamine, as the case may be. As Aspartic acid or Asparagine, as thecase may be. O-t-Bu--O-t-butyl DMF-Dimethylforrnamide orN,N-dimethylformamide DCCD-Dicyclohexylcarbodiimide SO H-Benzenesulfonicacid CH SO Hp-T0luenesulfonic acid DMAC-Dimethylacetamide orN,N-dimethylacetamide i-BuO fi ClIsobuty1 chloroforniate (or is obut ylchlorocarbonate) l-BuO CO Cl-See i-BuO 1(1) 01 R.T.Room temperature Thefollowing paragraphs 1 to 15 set forth general methods and conditionsfor the examples hereinafter which are usually in abbreviated form:

(1) Mixed anhydride coupling reaction. The procedure consists of (1)forming a mixed anhydride by reacting an a-acylamino acid or ana-acylamino peptide with a lower alkyl chloroformate in the presence ofa tertiary amine base, and then (2) reacting the resulting mixedanhydride with an amino acid derivative having a free amino group or apeptide derivative having a free amino group, to form a peptide. Theprocedure is described generally in a review by Albertson, Noel F., inChapter 4, Synthesis of Peptides With Mixed Anhydrides, pages 157-355,Organic Reactions, Volume 12, John Wiley and Sons, Inc., New York,London, 1962. This general technique is applied in Examples 18, 27, 34,3 6, 50, 56, 58, 65, 67, 69, 75, 80, 102, and 104.

(2) Alkaline hydrolysis, or saponification of an ester, a fundamentalmethod of de-esterification described by Fieser, Louis F. and Fieser,Mary, Organic Chemistry, Third Edition, Reinhold Publishing Corporation,New York, 1956, on page 178. This general technique is ap plied inExamples 19, 37, 66 and 68.

(3) Acid cleavage of an amino-blocking t-butyloxycarbonyl group, aconventional technique of peptide chemistry, described by Schrtider,Eberhard and Liibke, Klaus, in (I) Amino-Protecting Groups, pages 3-51of Volume 1, Methods of Peptide Synthesis, of The Peptides, AcademicPress, New York and London, 1965. This general technique is applied inExamples 20, 22, 24, 26, 30, 32, 39, 42, 47, 49, 53, 55, 57, 63, 70, 81,86, 88, 95, 97, 101, 103 and 105.

(4) Coupling of an N-hydroxysuccinirnide ester of an amino acidderivative or of a peptide derivative with an amino acid derivative orwith a peptide derivative, a technique described by Anderson, G. W., etal., Journal of The American Chemical Society, volume 86, pages1839-1842 (1964); ibidem, volume 85, page 3039; Kisfaludy, L., et al.,ACTA Chemica Academiae Scientiarum Hungaricae, Tome 44, Fascicles 1-2,pages 33-35 (1965); and Low, opere citato, pages 61-66 (196 5). Thisgeneral technique is applied in Examples 21, 23, 25, 31, 33, 35, 46, 54,64, 79, 87, 89, 94, 96, 98, 106.

(5) Coupling by use of WoodWards reagent, a technique describedgenerally by Schriider, Eberhard and Liibke, Klaus in (111) Formation ofthe Peptide Bond, pages 113-114 of volume 1, Methods of PeptideSynthesis, of The Peptides, Academic Press, New York and London, 1965.This technique is applied in Examples 28, 41, 43, and 48.

(6) Cleavage of a p-nitrobenzyl ester by catalytic hydrogenolysis, aconventional technique in organic and peptide chemistry, describedgenerally by Schrtider, Eber- 7 hard and Liibke, Klaus in (11)Carboxyl-Protecting Groups, pages 61-62 of volume I, Methods of PeptideSynthesis, of The Peptides, Academic Press, New York and London, 1965.This general method is applied in Examples 29, 44, 51, 59 and 61.

(7) Cleavage of a benzyloxycarbonylamino acid or of abenzyloxycarbonylamino peptide by catalytic hydrogcnolysis, aconventional technique of peptide chemistry, described by Schrider,Eberhard and Liibke, Klaus, in (1) Amino-Protecting Groups, pages 22-30of volume I, Methods of Peptide Synthesis, of The Peptides, AcademicPress, New York and London, 1965. This general technique is applied inExample 76.

(8) Coupling by use of dicyclohexylcarbodiimide, a procedure describedby Schrtider, Eberhard and Liibke, Klaus in (111) Formation of thePeptide Bond, pages 108-111, of volume 1, Methods of Peptide Synthesis,of The Peptides, Academic Press, New York and London, 1965. Thistechnique is applied in Examples 38, 52, 60, 62, 77, 82, 84 and 85.

(9) Neutralization of an acid addition salt, an elementary conversion ofchemistry, neutralization being defined on page 568 in the ThirdEdition, Hackhs Chemical Dictionary, McGraw-Hill Book Company, New York,Toronto, London, 1944. This technique is applied in Example 40.

(10) Formation of an N-hydroxysuccinimide ester of an amino acid or of apeptide. Either the mixed anhydride method (see (1), hereinabove) orcoupling by use of dicyclohexylcarbodiimide (see (8), hereinabove) maybe used. This technique is applied in Examples 45 (DCCD), 90, 91, 93, 99(MA) (11) Conversion of an ester or a mixed anhydride to an amide byreaction of the ester with ammonia, on elementary organic chemistrytransformation described by Fieser, Louis E, and Fieser, Mary, OrganicChemistry, Third Edition, Reinhold Publishing Corporation, New York,1956 on pages 178-180, 445-446. This technique is applied in Examples69, 71, and 80.

(12) Stripping of all blocking groups in a peptide (--S benzyl and Obenzyl ether groups from cysteine and serine, Nnitro blocking groupsfrom arginine amino acids, and t-butyloxycarbonyl N-terminalaminoblocking groups) by use of hydrofluoric acid. The technique hasbeen described by Sakaribara, S., et al., Bulletin of the ChemicalSociety of Japan, vol. 40, pages 2164, etc., (1967); ibidem, volume 41,pages 438-441 (1968); ibidem, volume 41, pages 1477-1479 (1968). Thistechnique is applied in Examples 72, 73, 74, and 107. (13) Acid cleavageof a t-butyl ester of a C-terminal peptide, a conventional technique ofpeptide chemistry, described in general terms by Schriider, Eberhard andLiibke, Klaus, in (11) Carboxyl-Protecting Groups, at pages 57-59, ofvolume 1, Methods of Peptide Synthesis, of The Peptides, Academic Press,New York and London, 1965. This general technique, but using acetic acidsaturated with hydrochloric acid as the cleaving medium, is applied inExample 78.

(14) Formation of t-butyloxycarbonylamino acid by use of t-butyl azidoformate, a technique described generally by Schriider, Eberhard andLiibke, Klaus in (1) Amino-Protecting Groups, pages 37-38 of vol. I,Methods of Peptide Synthesis, of The Peptides, Academic Press, New Yorkand London, 1965. This technique is applied in Example 92.

(15) Reaction of an N-hydroxysuccinirnide ester of a peptide withammonia to form a peptide amide is described in Example 100.

DETAILED DESCRIPTION The following examples illustrate in detail thepreparation of peptides by the use of mixed anhydrides and the peptidesresulting therefrom.

8 EXAMPLE 1 Preparation of benzyloxycarbonylglycyl-L-phenylalanylglycine ethyl ester -15 C. to R.T. Z-Gly-Phe-OH HGIYOEtThe mixed anhydride is prepared by adding 1.78 g. ofcarbobenzoxyglycyl-L-phenylalanine (0.0050 mole) to the reaction vesseland adding 25 ml. of tetrahydrofuran. The solution is agitated and thetemperature of the contents lowered to '15 C. To this solution is added1.01 g. of N-methylmorpholine (0.010 mole), followed by the addition of0.67 ml. of isobutyl chloroformate (0.0050 mole). After a period of 12minutes 0.53 g. of ethyl glycinate (0.0050 mole) is added and thematerial is then allowed to warm to room temperature. The product isisolated by adding the material to water and collecting the product oralternatively, by removing almost all of the tetrahydrofuran underreduced pressure and dissolving the residue in ethyl acetate (or othersuitable solvent). The product is washed with appropriate reagents untila neutral wash is obtained. Evaporation of the solvent yields 2.15 g. ofthe tripeptide. Recrystallization from a 2% alcoholic solution gives2.06 g. of pure L-tripeptide (93.2%) and none of the Dlrisomer. Arepeated experiment gives a 93.4% yield of pure -L-isomer and noDL-isomer.

EXAMPLE 2 Preparation of benzyloxycarbonylglycyl-L phenylalanylglycineethyl ester 15 C. to R.T.

When Example 1 is repeated using N,N-dimethylpiperazine in place ofN-methylrnorpholine, the yield of pure L-isomer is 94%. No -DL-isomer isdetected. A duplicate experiment gives a 96% yield of L-isomer and noDL- isomer.

EXAMPLE 3 Preparation of benzyloxycarbonylglycyl-L phenylalanylglycineethyl ester Using the procedure of Example 1 and replacingtetrahydrofuran with ethyl acetate, the yield of -I.-isomer is 87% andno D'L-isomer is isolated.

EXAMPLE 4 Preparation of benzyloxycarbonylglycyl-L- phenylalanylglycineethyl ester -15 0. to R.I. Z-GlY-Ph0-OH Hoi ont Z-Gly-Ihe-GlyOEt Whenexample 1 is repeated using dioxolane in place of tetrahydrofuran, an89% yield of L-isomer and no DL-isomer is obtained.

EXAMPLE 5 Preparation of benZyloxycarbonylglyc'yl-L- phenylalanylglycineethyl ester When the procedure of Example 1 is repeated usingN,N,N,N'tetramethylaminobutyne-2 in place of N-methylmorpholine, theyield of pure L-isomer is 81.6%. No DL-isomer is isolated.

EXAMPLE 6 Preparation of benzyloxycarbonylglycyl-L- phenylalanylglycineethyl ester When Example 1 is repeated using fl-cyanoethyldimethylamine,the yield of pure L-isorner is 90.7%. No DL- isomer is isolated.

EXAMPLE 7 This example demonstrates that when a tertiary amine otherthan the special defined amines of this invention is used, even in 1:1equivalent amounts, racemization may occur.

To 1.78 grams (0.005 mole) of carbobenzoxyglycyl L- phenylalanine in a100 milliliter flask is added 25 milliliters of tetrahydrofurancontaining 0.70 milliliter (0.005 mole of triethylamine. The solution isstirred mechanically and cooled to -'l5 C. Then 0.70 milliliter (0.00525mole) of isobutyl chloroformate is added to the cooled solution. After aperiod of 12 minutes 0.54 gram (0.00525 mole) of ethyl glycinate isadded, and the mixture in stirred until room temperature is reached. Thecrude prod-- uct is isolated with the removal of nearly all of thetetrahydrofuran by applying reduced pressure.

The crude product above is dissolved in 75 ml. of ethyl acetate and thesolution is washed successively with 25 ml. portions of a sodiumbicarbonate solution, water, 1 N hydrochloric acid, and water. Thewashed ethyl acetate solution is dried with anhydrous sodium sulfate.Evaporation of the dried solution yields the crystalline tripeptitde.Recrystallization of this material from ethanol (1 part of product to 50parts of solvent) yields 1.86 grams (84.5%) of pure L-isomer. About 181milligrams (8.2%) of 'D L-isomer was isolated. A duplicate experimentgives 79% of L-isomer and 7.2% of DL-isomer.

EXAMPLES 8-15 When two molar equivalents of the following amines areused in the procedure of Example 7, a common procedure in the prior artwhen the hydrochloride of the amino acid ester is used, the followingresults are obtained:

Yield, percent Attempted preparation of BOO-Arg-Asn-Leu-ONB (L, L, L),t-Butyloxycarbonyl-N-nitro-L-arginyl-If?sparag1nyl-L-leucine-p-11itrobenzyles er A one molar equivalent of r BOC'Arg-OH t-butyloxycarbonyl Nnitro-L-arginine and one molar equivalent of N-methylmorpholine isdissolved in tetrahydrofuran with warming. The resulting solution iscooled to about 0 C., and one molar equivalent of isobutyl chloroformateis added.

A separate solution of one molar equivalent of "EPA xH Asn Leu ONB,L-asparaginyl-L-leucine-p-nitrobenzyl ester trifiuoroacetate and onemolar equivalent of triethylamine in tetrahydrofuran is prepared atabout 0 C. This latter solution is then added to the pre-formed mixedanhydride mixture.

10 At least four neutral compounds are finally isolated, one of which isidentified as the diketopiperazine ofL-asparaginyl-L-leucine-p-nitrobenzyl ester. The desired tripeptidecould not be isolated.

EXAMPLE l7 Attempted preparation of BOC-Ser-Ala-Tyr-ONB (L,L,L),t-butyloxycarbonyl-L-seryl-L-alanyl-L-tyrosine p-nitrobenzyl esterHCLxH-AIa-Tyr-ONB-i-BOCuSer'OH BOC-Ser -Ala- ONB One molar equivalent ofBOC-Ser-OH, t-butyloxycarbonyl-L-serine, is dissolved in tetrahydrofurancontaining one molar equivalent of N-methylmorpholine, and the resultingsolution is chilled to about l3" C. To this chilled solution one molarequivalent of isobutyl chloroformate is added.

When an attempt is made to prepare a solution of the free baseH-Ala-Tyr-O'NB by neutralizing the with one equivalent of sodiumbicarbonate, the effort is frustrated because of instant formation ofcrystalline diketopiperazine, melting at 283286 C.

EXAMPLE 18 Preparation of t-but'yloxycarbonyl-L-valyl-L-leucinep-nitrobenzyl ester Preparation of t-butyloxycarbonyl-L-valyl-L-leucineR.T. BOC-Val-LeuONB BOC-Va1-Leu-OH(L,L)

EXAMPLE 20 Preparation of L-valyl-L-leucine trifluoroacetate EXAMPLE 21Preparation of t-butyloxycarbonyl-S-benzyl-L-cysteinyl-L-valyl-L-leucine EXAMPLE 23 Preparation oft-butyloxycarbonyl-L-threonyl-S-benZyl-L- cysteinyl-L-valyl-L-leucineSBzl ] RT. BOC-Tlir-OSu TFAxH-Cys-Val-Leu-OH SBzl BOC'ThT-(JYS-VEI'LOH-OII (L,L,L,L)

EXAMPLE 24 Preparation of L-threonyl-S-benzyl-L-cysteinyl-L-valyl-L-leucine trifluoroacetate 13 14 1 C R EXAMPLE 40 5 .t .T. Z2.HiS.0HH.Arg-Me zi-flis-lir -oMe Preparation of L-histidyl-N-nitro-L-arg1nyl-L-phenylalanine p-nitrobenzyl ester To 400 millilitersof tetrahydrofuran in a one-liter flask N0 equipped w1th a mechanicalstirrer was added 33 grams 5 2 N02 (0.075 mole) ofdibenzyloxycarbonyl-L-histidine and 18.2 NQHOOS milliliters ofN-methylmorpholine. The mixture was cooled to about -1s 0. using amethanol-ice bath. 10.05 milli- EXAMPLE 41 liters of isobutylchloroforrnate was added with rapid Preparation oft-butyloxycarbonyl-L-asparaginyl-L- stirring and the reaction formingthe mixed anhydride was phenylalanine p-nitrobenzyl ester allowgdtohciimtinue for 4 minutes. At the end of this EXAMPLE 42 perio a c i edsuspension of 20.18 grams (0.075 mole) of the methyl nitroargininehydrochloride and 10.5 milli- Prepmim 2 f gffl fifY liters (0.075 mole)of triethylamine in 200 milliliters of Zy e y e dimethylacetamide wasadded. The suspension was itself prepared by dissolving methylnitroarginine hydrochloride 2 BOC'ASH'PMONB +1101 HCIZH'ASHPMONB (LL) indimethylacetamide, warming, adding triethylamine and 0 [EXAM LE 43cooling; triethylamine hydrochloride precipitates on 0001- P ing. Thereaction mixture with the chilled suspension Preparation of tbutyloxycarbonyl L asparaginyl L- added was stirred cold for two minutesand further stirred asparaginyl-L-phenylalanine p-nitrobenzyl ester R.T.HClxH'Asn-PheONB BOC-Asn-OH BOC'Asn-Asn-Phe-ONB (L,L,L)

+reagent K at room temperature for about 40 minutes. The mixture EXAMPLE44 was then filtered. The filtrate was concentrated under reducedpressure to an orange colored, oily residue. The Preparation of t-buty yy p r g y -L- residue was dissolved in about 300 milliliters of ethylasparaginyl-L-phenylalanine C. to 50C. BOC-Asn-Asn'Phe-ONBBOC-Asn-Asn-Phe'OH (L,L,L)

H2, Pd

acetate and washed successively with two 100 milliliter M portions ofwater, two 100 milliliter portions of dilute EXA PLE 45 sodiumbicarbonate and finally with 250 milliliters of Prepq of y Q yCab0ny1L-asparag1nyl-L- dilute sodium chloride. The organic phase wasdried over a p r gmyl-L-phenyl lamne N-hydl'oxysucc mmlde ester 0 0. to5 C. BOC-Asn-Asn-Phe-0H BOC-Asn-Asn-Phe-OSu (L,L,L)

DooD+H0su anhydrous sodium sulfate and filtered and concentrated EXAMPLE46 under reduced pressure to a pale yellow oil. The strong P b v 45reparation of t-butyloxycar ony -aspara g1nyl-L-asparorange c.0101-removed. In the t Wash T aginyl-L-phenylalanyl Lhistidyl-NG-nitro-L-arginylyellow 011 was triturated wlth ether w1thvigorous rubbing bPhenYlalanine pmflmbenzyl ester to yield an amorphousproduct. Yield 41.5 grams (87%) of theoretical. 11102 EXAMPLE 37 50BOC-Asn-Asn-Phe-OSu H-His-Arg-Phe-ONB N02 Preparation of f gi fjgggg igL hlstldyl N B0c-Asn-Asn-rhe-His-iirg-Phe-oNB (L,L,L,L,L,L)

EXAMPLE 47 NO Preparation of L- asparaginyl L- asparaginyl-L-phenyliR.l. alanyl L histidyl NG nitro-L-arginyl-L-phenyl- Tg'OMB 16% (LIL)alanine p-nitrobenzyl ester N02 A R.T. BOO-Asn-Asn-Phe-His g-Pno-ONB WEXAMPLE 38 a 3 Preparation of henzyloxycarbonyl-L-histidyl-N -nitro-H-Asn-Asn-Phe-His-Arg-Phe-ONB (L,L,L,L,L,L)

L-arginyl-L-phenylalanine p-nitrobenzyl ester .T. Z-His-rirg-OHqhsoaHrH-Phe-oNB Z-His-iirg-Phe'ONB (L,L,L)

DOCD+HOSu .T. Z-His-Airg-Phe-ONB 2HBt H-HisALrg-Phe-ONB (L,L,L)

HBr HOAc 1 5 EXAMPLE 4s EXAMPLE 52.

Preparation of t-butyloxycarbonyl-L-asparaginyl-L-leucine DmitrobenzYlester Preparation of t-butyloxycarbonyl NG nitro-L-arginylreagent KEXAMPLE 49 Preparation of L-aspara ginyl-L-leucine p-nitrobenzyl L- i 1L 1 1 L asparaginyl L asParaester tnfluoroacetateginyl-L-phenylalanyl-L-histidyl NG nitro-L-arginyl- R.T. L-phenylalaninep-nitrobenzyl ester BOC-ASI1-Lel1-ONB TFAzH'Asn-Leu-ONB (L,L)

+ TFA B OC-Airg-Asn-LewOH H-Asn-Asn-Phe-His-iirg-PlleONB DCCD HOSu NO; N02 B O CJirg-Asn-LeuAsn-Asn-Phe-His-Arg-Phe- ONB (L,L,L,L,L,L,L,L,L)

EXAMPLE 50 N 02 Preparation of B00 Arg-Asn-Leu-OBB (L,L,L),t-butyloxycarbonyl-NG, nitro-L-arginyl-Irasparaginyl-L-leucinep-nitrobenz yl ester l M.A. BOC-Arg-OH TFAzH-Asn-Leu-ONBBOO-JLrg-Asn-Leu-ONB C. to 0 C.

A 3.80 gram (0.0139 mole) portion of EXAMPLE 53 N02 Preparation ofNG-nitro L arginyl L asparaginyl-L- @011 leucyl L asparaginyl Lasparaginyl L phenyl- (t-butyloxycarbonyl NG nitro L arginine) and 3.06i ig mtro L argmyl'L'phenyl' milliliters (0.0278 mole) ofN-methylmorpholine were a 2mm: p'mtro enzy ester mixed in about 150milliliters of tetrahydrofuran and f warmed on a steam bath. Dissolutionwas incomplete. The Ayg. A .Ph .Hi .A .1 h .ONB small amount ofinsolubles was separated by filtration. 110M, H01 The filtrate wascooled to -20 C. in a Dry Ice-acetone gs e hee bath. A 1.86 milliliter(0.0139 mole) portion of iso-butylchloroformate was added and the mixedanhydride formed EXAMPLE 54 in 4 minutes. A solution of 6.88 grams(0.0139 mole) of Preparation of t-butyloxycarbonyl L tryptophanyl-NG-TFAxH-Asn- Leu-ONB, L asparaginyl L leucine pnitro-L-asparaginyl Lleucyl L asparaginyl-L-asnitrobenzyl ester trifluoroacetate, in 40milliliters of paraginyl L phenylalanyl L histidyl NGnitrotetrahydrofuran was added and the mixture was removedL-arginyl-L-phenylalanine p-nitrobenzyl ester rlvol lilo: R TBOC-Try-OSu H.Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-ONB 1Y0; N02BOC-Try-Arg-EAsn-Leu-Asn-ASnPhe-His-Arg-Phe'ON B (L,L,L,L,L,L,L,L,L,L)from the bath and placed in a refrigerator at 0 C. over- EXAMPLE 55night. The mixture was then concentrated in vacuo and 50 the residue wasextracted into a mixture of 15 0-200 milli- Preparation ofL-tryptophanyl N nitro L arginyl-L- liters of ethyl acetate and 50milliliters of water. The asparaginyl L leucyl L aspara'ginyl Lphenylethyl acetate layer Was separated and washed successivelyalanyl-L-histidyl N nitro L arginyl L phenylwith 50 milliliter portionsof dilute phosphoric acid, water, alanine p-nitrobenzyl ester I lBOC-Try-Arg-Asn-Leu-Asn-Asn-Phe-His-Arg-Phe-ONB TFA, NaHCOz 11102 Y ZH'TryArg-Asn-Leu-Asn-Asn-Phe-Hls-ArgPhe-ONB (L,L,L,L,L,L,L,L,L,L) 10%sodium bicarbonate solution, and finally water. The EXAMPLE 56 washedethyl acetate layer was dried over sodium sulfate and concentrated invacuo to yield a gum. This gum was Preparatlon of t-butyloxycarbonyl Lalanyl L tryotriturated with diethyl ether and Washed from the flask smep-nitro ester BOC'AlaOH CHaOSOaHIH,Tyr-ONB I BOC-Ala-Tyr-ONB (L,L)

-12 c. to 20 0.

with diethyl ether to give 8.31 grams (91% yield) of an A solution wasmade of 9.45 grams (0.050 mole) of amorphous solid. the BOC-Ala-OH,t-butyloxycarbonyl L alanine, and EXAMPLE 51 11.0 milliliters (0.100mole) of N-methylmorpholine in 50 milliliters of tetrahydrofuran. Thissolution was chilled Prepamm to -12" c., and 6.70 milliliters 0.050mole) of iso'butyl asparagmyl-L'leucme chloroformate was added. After 60seconds (activation 11:02 R T 7 time) this chilled solution was added toanother solution, BOC.A -g.A5n-Leu-ONB BOC-Arg.Asn.Leu-OH L,L,L whichhad been prepared immediately beforehand and Pd was at 20 C. at the timeof addition, of 24.42 grams (0.050 mole) of CH SO HxH-Tyr-ONB,L-tyrosine pwas filtered to remove insoluble material contained in thenitrobenzyl ester p-toluenesulfonic acid salt, in 100 milliproduct. Thesolution was cooled to 13 C. and 0.67 liters of dimethylformamide. Theresulting mixture Was milliliters (5 millimole) of isobutylchloroformate was left overnight at C. added. Thirty seconds later, asolution of 2.12 grams 5 millimoles) of HClxH-Ala-Tyr-O'NB(L-alanyl-L-tyro- The next day the reaction mixture was added to 1 litersine p-nitrobenzyl ester hydrochloride) in milliliters of of water, andthe resulting mixture, a gummy precipitate tetrahydrofuran at 5 C. wasadded. The mixture Was and supernatant water, was refrigeratedovernight. stirred for one hour. The product was isolated as a gum Thenext day (the gum not having crystallized) the and dried. Thin layerchromatography of the product using supernatant water was decanted andthe gummy residue 10 the solvent system n-butanolzacetic acidzwater(40:4:12)

was dissolved in 500 milliliters of ethyl acetate. was used to followthe reaction. The sharp diminution of The ethyl acetate solution wassuccessively washed with spot intensity at Rf 0.58 (HClxH-Ala-Tyr-ONB)and water, aqueous sodium bicarbonate solution and then emergence of astrong spot from R 0.73 to 0.92 indicated again with water. The washedethyl acetate solution was the reaction to be essentially complete. thendried over anhydrous sodium sulfate. 1

Thin layer chromatography using the system 95 parts EXAMPLE 59 ethylacetate: 5 parts ethanol showed a heavy ultra violet Preparation oft-butyloxycarbonyl-L-seryl-L-alanyl-L positive spot at the front, andthe same very heavy spot tyrosine at the front positive totolidine-chlorine. There was a faint R.T. tolidine-chlorine positivespot at R 0.40. BOC-Ser-Ala-Tyr-ONB BOC-Ser-Ala-Tyr-OH (L,L,L)

The ethyl acetate solution was evaporated in an open dish, leaving ayellow oil. The oil was dried by applying EXAMPLE 60 a low vacuum,leaving a cotton candy residue. The Preparatwn 0f y y y y y y finalyield of dried product was 21.5 grams, 91.4% of Y yp p y 1mm Larginyl-L35 the theoretical yield. paraginyl leucyl asparaginyl L-phenyl- Thin layer chromatography using the system 8 parts nalanyl Lh1st1dyl N nitro L arginyl L phenylheptane: 2 parts isopropanol gave aspot at R: 0.47 posil e p-nl r benzyl ester N02 11102 R T 130 Q.se-.A1a,.Tyr.OH H-Try-Arg-Asn-LewAsn-Asn-Phe-His-Arg-Phe-ONB NO: N 02 3.sm .iirg-Asn-Leu-Asn-Asn-Phe-HisArg-Phe-ONB (L,L,L,L,L,L,L,L,L,L,L,L,L)tive to both ultra violet and tolidine-chlorine, but negative EXAMPLE 61to mnhydnn' Preparation of t-butyloxycarbonyl L seryl L alanyl- Theabove reaction was repeated, and the product was LJYIOSYI L tryptophanylL nitro L arginyL dried under high vacuum over phosphorus PGIilZOXIdB.The L asparaginyl L leucyl L asparaginyl L aspara yield was 24.2 grams,substantially quantltative, meltlng ginyl L phenylalanyl L histidyl N at7072 arginyl-L-phenylalanine III-O2 7 2 R T 1300.3 A1;Tyr-Try-Arg-Asn-Leu-Asn-Asn-PheHiS-Arg'Phe-ONB H P d NO: NO: B OG-Ser-Ala- Tyr-Try-Lrg-Asn-Leu'Asn-Asn' Phe-His-Lrg-Phe- OH(L,L,L,L,L,L,L,L,L,L,L,L,L) EXAMPLE 57 EXAMPLE 62 Preparation oft-butyloxycarbonyl L seryl L alanyl- L-tyrosyl L tryptophanyl N nitro Larginyl-L- asparaginyl L leucyl L asparaginyl L aspara- R T ginyl Lphenylalanyl L histidyl N nitro L- BOC-Ala-TyrONB I-IClIH-Ala-Tyr-ONB(L,L) arginyl L phenylalanyl O benzyl L -serylglycyl- L-methinonineamide Preparation of L-alanyl-L-tyrosine p-nitrobenzyl esterhydrochloride IIIOZ NO; (|)Bzl R TBOG-Ser-Ala'Tyr-Try-Arg-Asn-LewAsn-Asn-Phe-His-Arg-Phe-OHTFAzH-Ser-GlyMet-NH;

DOCD H0811 IIIO: N02 (1)1321 BOC-Ser-Ala-Ty Y- g'Sn-Phe-His-Arg-Phe'SerGly-Met-NH2 (L,L,L,L,L,L,L,L,L,L,L,L,L,L,L)

EXAMPLE 58 Preparation of BOC-Ser-Ala-Tyr-ONB (L,L,L),t-butyloxycarbonyl L seryl L alanyl L tyrosine p- 60 EXAMPLE 63mtro'benzyl ester Preparation ofL-seryl-L-alanyl-L-tyrosyl-L-tryptophenyl- O OH to E; N -nitro L arginylL asparaginyl L leucyl-L- HCM'H'AMTYI'ONB Bo asparaginyl L asparaginyl Lphenylalanyl L- M.A. BOC'Ser'Ah'TYr'ONB 65 histidyl N nitro L arginyl Lphenylalanyl-O- A 2.05 gram (5 millimole) portion of BOC-Ser-OH benzyl Lserylglycyl L methionine amide trifluoro- (t-butyloXycarbonyl-L-serine)and a 1.10 milliliter (1O acetate millimole) portion ofN-methylmorpholine were added to 20 milliliters of tetrahydrofuran andstirred. The solution 1 9 EXAMPLE 64 Preparation oft-butyloxycarbonyl-S-benzyl-L-cysteinyl-O- benzyl L seryl L asparaginylL leucyl O- benzyl L seryl L threonyl S benzyl L cysteinyl-L-valyl Lleucyl L seryl L alanyl-L-tyrosyl- L-tryptophanyl N nitro L arginyl Lasparaginyl L leucyl L asparaginyl L asparaginyl-L- phenylalanyl Lhistidyl N nitro L arginyl-L- phenylalanyl O benzyl L serylglycyl Lmethionine amide OBzl (tetrahydrofuran) above, and the resultingsolution was allowed two minutes of activation time at 13 C., afterwhich the two solutions were combined. The resulting mixture was allowedto stand overnight.

The next morning the solvent tetrahydrofuran was removed under vacuum,and the residue was then shaken into a mixture of 75 milliliters ofethyl acetate and 50 milliliters of water. The resulting ethyl acetatelayer was separated and then Washed successively with dilute 10 aqueousphosphoric acid, water, dilute aqueous sodium N02 N0 0 B21 SBzl OBzl01321 81321 I l 130 CCys-Ser-Asn-Leu-Ser-Thr'Oys-Val-Leu-Ser-Ala-Tyr-T13;-Arg-Asn-Leu-AsnAsn-Phe-His-Arg-EXAMPLE 65 Preparation of t-butyloxycarbonyl-O-benzyl-L- serylglycinemethyl ester OBzl 01321 EXAMPLE 66 Preparation oft-butyloxycarbonyl-O-benzyl-L- serylglycine OBzl OBzl RJI. BOQSer-GIy-OMe BOC-ISGI-GIY-OH (L) NaOH EXAMPLE 67 l Preparation ofBOC-Ser-Gly-Met-OMe (L,L),tbutyloxycarbonyl-O-benzyl-L-serylglycyl-L-methionine methyl ester OBZIOBzl I BOC-Ser-Gly-OH+ HClxH-Met-OMQ BOC-Ser-GlyMet-OMe 2.40 grams(0.007 mole) of OBzl l B O O'SeuGly-OHt-butyloXycarbonyl-O-benzyl-L-serylglycine, was dissolved in a solutionof 1.54 milliliters (0.014 mole) of N- methylmorpholine in millilitersof tetrahydrofuran.

Another solution was prepared of 1.4 grams (0.007 mole) ofI-I'ClxH-Met'OMe, L-methionine methyl ester hydrochloride in 5milliliters of dimethylacetamide.

Then, 0.93 milliliter (0.007 mole) of isobutyl chloroformate was addedto the first of the two solutions 51321 01321 OlSzl Slizl N0 bicarbonatesolution, and water. The washed ethyl acetate solution was then driedover anhydrous sodium sulfate. Thin layer chromatography on silica ofthis ethyl acetate solution using the solvent system 1 part isopropanol:

l BOO-Ser- Gly-OMc (L) 1 part n-heptane gave a spot at R 0.74 positiveto ultra violet (very strong), positive to tolidine-chlorine (heavy,brown-yellow) and positive for divalent sulfur (very strong).

The ethyl acetate was evaporated, first under low 5 vacuum, then pumpvacuum, leaving 3.40 grams of product, equivalent to a 97% yield.

EXAMPLE 68 Preparation of t-butyloxycarbonyl-O-benzyl-L-serylglycyl-L-methionine BO C-Ser- Gly-Met-OH (L,L) aOH EXAMPLE 69Preparation of t-butyloxycarbonyl-O-benzyl-L- serylglycyl-L-rnethionineamide OBzl OBzl --l0 C. to RIP. BOC-SenGly-Met'OH BOO-Scr-Gly-Mct-Nll(L,L

M.A., NH;

EXAMPLE 70 Preparation of O-benzyl-L-serylglycyl-L- methionine amidetrifiuoroacetate OBzl OBzl R.T. BOC-Ser-Gly-Met-NHzTFAzI-I-Ser-Gly-Met-NH (L,L)

+TFA

RIP."

| I IBOC-Cys-SouAsu-LewSeuThr-Cys-Val-Luu-Ser-Ala-Tyr-Try-Arg-Asu'Lou-Asu-Asn-Phe-His-Arg-27 EXAMPLE 99 Preparation of t butyloxycarbonyl-N -nitro-L-arginyl-L-asparaginyl-L-leucine N-hydroxysuccinimide ester Preparation oft-butyloxycarbonyl-N -nitro-L-arginyl-L- asparaginyl-L-leucine amideEXAMPLE 101 Preparation of N -nitro-L-arginyl-L-asparaginyl-L- leucineamide trifluoroacetate EXAMPLE 102 Preparation of ECG Ser -Ala -Tyr -Try-Arg Asn Leu NH (L,L,L,L,L,L,L), t-butyloxycarbonyl-O-benzyl-L- seryl Lalanyl-L-tyrosyl-L-tryptophanyl-N -nitro-L- arginyl Lasparaginyl-L-leucine amide N02 C. to C.

(I) B 21 ITTO z B O C'Ser'Ala-Tyr-Try-OH TFAzH-Arg-Asn-Leu-NH A solutionof 1.24 grams (1.54 mmoles) of BOC-Ser-Ala-Tyr'Try-OHt-butyloxycarbonyl-O-benzyl-L-seryl L-alany1 L tyro syl-L-tnyptophane,in milliliters of tetrahydrofuran was stirred and chilled in a 15 C.bath. Then 0.35 milliliter (3.08 mmoles) of N-methylmorpholine wasadded, followed Within a few minutes by addition of 0.21 milliliter(1.54 mmoles) of isobutyl chloroformate.

An immediate precipitate formed. After 1 minute a solution of 0.86 gram(1.54 mmoles) of the TFA salt, N nitro-L-arginyl L-asparaginyl-L-leucineamide trifluoroacetate, in 5 milliliters of dimethylformamide was added(using an additional 1 milliliter of dimethylformamide as a rinse). Theresulting solution was neutral (vapors) to general purpose pH paper,and, so, another equivalent (0.17 milliliter) of N-methylmorpholine wasadded, after which the vapors were basic. After 2 or 3 minutes thecooling bath was removed, and the reaction mixture was stirred for anadditional 10 minutes. At this point, and also several hours later thanlayer chromatography of small aliquots of the reaction mixture on silicausing the 40:20:4 solvent system showed about the same picture:strongest spot at R; 0.8, positive to ultraviolet, Ehrlichs OBZl N02 1ywith several small portions of water, giving a yellow solid, batch A.Thin layer chromatography of this material on silica using the 40:20:4solvent system and spotting the samples in dimethylformamide gave astrong spot at R, 0.8, positive to ultraviolet, Ehrlichs reagent, andtolidine-chlorine (2 plates); and faint similar spots at R;

0.95 and 0.6; a head on the R; 0.8 spot detected by Ehrlichs reagentonly; and a faint spot positive to ultraviolet only at R 0.2. Thetetrahydrofuran-dimethylformamide filtrate showed the same spots, butthe impurities were stronger. Consequently the filtrate was concentratedusing a vacuum evaporator, and the resulting concentrate was treatedwith water and hydrochloric acid, and chilled, giving additional solid.This latter material was collected by filtration, washed with water andN aqueous sodium bicarbonate solution, and then again with water, batchB. Batch B was more strongly colored than batch A, but by thin layerchromatography showed 0 B21 IIIOZ B O CBer-AlaTyr-Try-Arg-Asn-Leu-NH,

the same major spot at R 0.8, and trace spots at R 0.6, 0.4(tolidine-chlorine positive), and 0.95. Both A and B were driedovernight in a vacuum oven, weights: A, 0.88 gram; B, 0.86 gram.

B was stirred with a few milliliters of ethyl acetate, dissolving muchof the color and causing the solid to become gelatinous. The solid wasseparated by filtration, and washed. The remaining solids were finallywashed with methanol, and then dried, batch A. Addition of ether to themethanol wash gave a little more solid, batch A". Thin layerchromatography of both A and A" gave the R, 0.8 spot with the head line.A and A", after drying, were combined with A, making batch B, a total of1.22 gram.

Finally a small additional amount of solid product was batch C. Thisproduct (0.19 gram) was combined with recovered from the methanol-etherfiltrate from A", the 1.22 grams of B, making a total of recoveredproduct of 1.40 grams (79% of the theoretical 1.77 grams).

EXAMPLE 103 Preparation of O-benzyl L seryl-L-alanyl-L-tyrosyl-Ltryptophanyl-N -nitro L arginyl-L-asparaginyl L- leucine amidetrifluoroacetate OBZl NO;

reagent, and tolidine-chlorine (separate chromatographic plate); minorspots at R, 0.94 and 0.6; an additional minor tolidine-chlorine spot atR; 0.7.

The considerable amount of solid present, somewhat gelatinous, wasseparated by filtration, and then washed (with difficulty) withtetrahydrofuran, and then separate OBzl ZBzl 0321 NO:

EXAMPLE 104 Preparation of t butyloxycarbonyl-O-benzyl-L-seryl-L-threonyl-S-benzyl-L-cysteinyl-L-valyl L leucyl O- benzylL-seryl-L-alanyl L tyrosyl L tryptophanylvN-nitro-L-arginyl-L-asparaginyl-L-leucine amide 29 30 EXAMPLE 105 2. Aprocess according to claim 1 in which the tertiary Preparation ofbenzyl-L-seryl-L-threonyl-S-benzyl-L- amme 1SN'methylmirPhdmecysteinyLL-valyl-L-leuoyl-O-benzyl-L-seryl L alanyl- 3.A process according to claim 1 1n Which the tertiary L-tyrosyl Ltryptophanyl N nitro L arginyl-L amine is y p p asparaginyl-L-leucineamide 4. In a process for the synthesis of peptides of natural- B OC-Ser- Thr- Oys-Val-Leu- S tar-AlaTyr-Try-rirg-Asn Leu'NHz TFA NaHOO;OBzi %Bzl |OBzl N02 H-enlhr-Cys-Va1-Leu-Ser-Ala-Tyr-Try-Airg-Asn-Leu-NEM(L,L,L,L,L,L,L,L,L,L,L,L) EXAMPLE 106 ly occurring a-amino acids by themixed anhydride meth- Preparation of tbutyloxycarbony1-Sbenzyl-L-cysteinyl- 0d wilereman blocked wammo acld orImptlde of lObenzy1 L seryl L aSparaginy1 .L 1eucy1 O benzylqf a-amrnoacids m which the block groups are benzyloxyseryl-L-threonyl-S-benzyl Lcysteinyl L valyl L- Carbonyl, tertlafybutyloxycafbOIIYL p y yleucyl-O-benzyl-L-seryl-L-alanyl-L-tyrosyl L trypt fluoroacetyl orphthaloyl and in which other reactive phanyl-N-nitro=L-arginy1-L-asparaginy1 L leu ine groups are blocked, is reactedwith an alkyl chloroamide formate at temperatures between about 5 C. and

S321 01321 01321 ZBzl 0321 N0 BOG-Cys-Ser-Asn-Leu-OSuH-enThnOys-Va1'Leu-Ser-Ala-Tyr-Try-irg-Asn-Leu-NHZ ZBzl OBzl OBzl ZBzlOBzl N02 BOO-Cys-Ser-Asn-Leu-SeIThriJys-VaLLeu-Ser'Ala-Tyr-Try-Arg-Asn-Leu-NHz(L,L,L,L,L,L,L,L,L,L,L,L,L,L,L, L) EXAMPLE 107 about C. for from about/2 minute to about 15 Preparation of L cySteiny1 L Sery1 L asparaginylminutes in the presence of a tertiary amine to produce a. leucyl L serylL threonyl L cysteinyl L valykb mixed anhydride and this anhydride isthen reacted with 1611cy1 L-Serypbalanypbtyr03y} L tryptophanyll, ana-amino acid or peptide having a free amino grouparginyl-L-asparaginyl-L-leucine amide hydrozfluoride and a protectedC-ter-minal carboxyl group to form a 81321 OBzl OBzl ZBzl OBzl N02 1L 600. to RT. BOC-Cys-Ser-Asn-Leu-Ser-TimCys-Val-Leu-Ser-AIaTyrTry'Ig-ASH'LGJI'N Z FHFxH-Cys-Ser-AsnLeu-Ser-Thr-Cys-Val-Leu-Ser'AlaTyr-Try-Arg-Asn-Leu-NH;(L,L,L,L,L,L,L,L,L,L,L,L,L,L,L,L) Iclaim: peptide, the improvement whichcomprises carrying out 1. In a process for the synthesis of peptides ofnaturalthe mixed anhydnde reaction by the use of a primary or lyoccurring u-amino acids by the mixed anhydride methsecondary lower alkylchloroformate and from 1 to about od wherein an amino blocked m-aminoacid or peptide of 2 equivalents of N-methylmorpholine in the presenceof a-amino acids, in which the blocking groups are benzyl- 40tetrahydrofuran.

oxycarbonyl, tertiarybutyloxycarbonyl, triphenylmethyl, References Citedtrifluoroacetyl or phthaloyl and in which other reactive UNITED STATESPATENTS groups are blocked, is reacted with an alkyl chloro 2,713 5747/1955 Vaughan 260 112 5 formate at temperatures between about 5 C. and2,927,107 3/1960 Velluz et a1- 260 112-5 about -20 C. for from about /2minute to about 15 minutes in the presence of a tertiary amine toproduce a 3264281 8/1966 Applewhlte et 260 112'5 mixed anhydride andthis anhydride is then reacted with OTHER REFERENCES an a-amino acid orpeptide having a free amine group Albertson Organic Reactions 12 172 179187495 and a protected C-terminal carboxyl group to form a (1962).

peptide, the improvement which comprises carrying out Anderson et a1. 1.Am. Chem 88 1338 1339 the mixed anhydride reaction with a primary loweralkyl (1966) chloroformate or secondary lower alkyl chloroformateAnderson et a1 Am Chem 89, ,5 7 and from 1 to about 2 equivalents of atertiary amine se- 19 7 lected from the group consisting ofN-methylmorpholine and N,N-dimethylpiperazine in the presence of a LEWISGOTTS Pnmary Exammer solvent selected from the group consisting oftetrahydro- M, KASSENQFF, A i t E i furan, dimethoxyethane,N,N-dimethylacetamide, ethyl acetate, dioxolane, triethylphosphate and5,5-dirnethyldioxolane. 260268 UK, 309, 326.14 T, 326.3, 463

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,640,99l Dated February 8, I979 Inventor(s) Fran-C18 Marc Callahan Itis certified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

ll Column 2, fomula NO, 2, please change H N,A -C-OS" to Column 10,Example 17, line 11, please change "BOC Ser-Ala-ONB" to readBOC-Ser-Ala'Tyr-ONB Column 15, Example 50, in the title, please changeOBB" to read ONB Co lumn 25, Example 89, line 32, please change ZBZlOBZl I SBZl OBZl "BOC- (J-ys -Se r .Asn-Leu-OSu" to read BOC-bys-Ser-Asn-Leu-OSu ZBzl OBzl Also, line 57, please change "BOG-Cys-Ser-Asn-Leu" to read 1321 pBzl BOC ys -Ser-Asn-Leu Column 28, Example10% line "I I, please change Q ZBzl OBzl SBzl BOG-:EBer-I'hrCys-Val-Leu"to read BOC-Een'lhnCys-Val-Leu Column 29, Example 105, line 7, pleasechange QBzl ZBzl QBzl SBzl "BOO-Ber.Thr.Cys.Val-Leu" to readBOC-Ser-Thr-Cys-Val.Leu

F Q po'wso USCOMM-DC 60376-P69 11.5. GOVERNMENT PRINTING O'Ilcl 1 I!"O-3-33l UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,6uo,991 Dated February 8, 1972 In veritofl s) Francis Marc ggllghan Itis certified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

Page 2 7 Column 29, Example 106 line 20, please change (31321 2321' )BzlSBzl I l T "H-Ser-Thr-Cys-Val-Lem to read H-Ser-Thr.Cys.Val -Leu Also,line 22, please change "BOC-Cys -Ser.Asn.Leu-Ser-Thr- ZBZl 1 S321 1 sCys -Val-Leu" to read BOG-Cys -Ser-Asn-Leu'Ser.Thr-( !ys .Val-Leu Column29, Example 107, line 29, please change 'Sl3zl OBzl O Bzl ZBzl"BOC-Cys.Ser-Asn-Leu-Ser-Thr-Cys" to read SBZl OBZl OBZl SBZl BOC- Cys-er-Asn-Leu-Ser-Thr-Cys Signed and sealed this 29th day-of August I972.

(SEAL) Attest:

EDWARD M .FLE'PCHER JR ROBERT GOTTSCHALK Attegting Officer Cnmmi q innp-n nf D a tpo'wso v USCOMM-DC scan-poo Q ":5. GOVINIINY PRINTINGOFFICE I x," -3-3$

